GSM6620143: RSLC1.; Escherichia coli; RNA-Seq
Bacterial cells were grown in 37°C, harvested at OD600 of ~0.6 and total RNA was isolated by trizol method. 4 μg of QC passed total RNA was used for ribo-depletion using RiboMinus Bacterial Kit (Invitrogen). Further, 100 ng of Qubit quantified ribo-depleted RNA was taken for Transcriptome library preparation according to Sure Select Strand-Specific RNA Library Prep Kit protocol outlined in “SureSelect Strand-Specific RNA Library Prep for Illumina Multiplexed Sequencing” (Illumina). Briefly, the RNA was fragmented for 4 minutes at 94oC in the presence of divalent cations and first strand cDNA was synthesised. The single stranded cDNA was cleaned up using High Prep (Magbio, Cat # AC-60050). Strand specificity was maintained by the addition of actinomycin D. Second strand cDNA was synthesised and end repaired using Second Strand Synthesis using End Repair mix. The cDNA was cleaned up using High Prep (Magbio, Cat # AC-60050). Adapters were ligated to the cDNA molecules after addition of “A” base. High Prep cleanup was performed post ligation. The library was indexed and enriched for adapter ligated fragments using 10 cycles of PCR. The prepared library was quantified using Qubit and validated for quality by running an aliquot on High Sensitivity Bioanalyzer Chip (Agilent). 150 (75X 2) bp Paired End Sequencing was carried out on Illumina platform to generate 20-25 million PE Reads per sample.